720
ANALYTICAL CHEMISTRY
sign or repair was made on the bomb. It is recommended that investigators determine their own constants and check these periodically. The technique described was developed for application to the microanalysis of biological matter where only minute samples are available. Emphasis was laid on the determination of linoleic and arachidonic acids, which are important components of animal material. The method does not appear to be useful for the analysis of acids more unsaturated than arachidonic, where another technique is of far greater advantage ( 5 ) . However, the method has been used successfully in the characterization of the lipides of liver particulate fractions ( 7 ) . LITERATURE CITED (1) Beadle, B. E-., and Kraybill, H. R., J . Am. Chem. S o c . , 66, 1232 (1944). (2) Bradley, T. F.. and Richardson, D., Ind. Eng. Chem.. 34, 237 (1942).
(3) Brice. B. A . , and Swain. AI. R., J . Optical SOC.Am., 35, 532 (1945). (4) Hilditch, T. P.,>lorton, R. A , . and Riley, J. P., Analus(, 70, 62 (1945). (5) Holman, R. T., and Burr, G . 0.. A r c h . B i o c h e m . , 19, 474 (1948). (6) Kass, J. P., Miller. E. S.. Hendrickson. J., and Burr, G. 0 . . Abstracts 99th Meeting ah. CHEM.SOC., p. 11, Cincinnati, 1940. (7) Kretchmer, N.,A r c h . Biochem. (in press). (8) Mitchell, J. H., Jr.. Kraybill, H. R., and Zscheile, F. P., IND. EKG.CHEM.,ANAL.ED., 15, 1 (1943). (9) hlowry, D.,Bmde, W.R., and Brown, J. B., J . Biol. Chem., 142, 671 (1942). (10) O'Connor, R. T.,Heinaelman, D. C., and Dollear, F. G., 01'1 & Soup, 22, 257 (1945).
R E C E I V E DAugust 8. 1949. P a r t of thesis presented b y Leah Cohadas t o t h e Graduate Faculty of t h e University of Minnesota in partial ftilfillrnent of t h e requirements for t h e M.S. degree. J u n e 1945. T h e work u a 8 aided by grants froni t h e S a t i o n a l Dairy Council on behalf of t h e Ameriran D a i r y Association and from the National Livestock a n d M e a t Board.
Microdetermination of Formaldehyde with Chromotropic Acid CLARK E. BRICKER AND W. AUBREY V A l L Princeton University, Princeton, N . J . The use of chromotropic acid as a specific reagent for determining formaldehyde has been extended 80 that it can be applied in the presence of large concentrations of various organic compounds. In the presence of chromotropic acid, these interfering organic compounds are removed b y evaporation, whereas the formaklehyde is retained by the reagent. Subsequent addition of sulfuric acid develops the true purple color due to the formaldehyde present.
I
S SEVERAL previous publications ( 2 , 3, i , 8) chromotropic acid, 1,8-dihydroxynaphthalene-3,6-disulfonic acid, has been suggested as a suitable reagent for a spectrophotometric method to determine small amounts of formaldehyde. More recently, Speck (10) found that diacetyl yields formaldehyde when heated with sulfuric acid and applied chromotropic acid to determine this compound. Boos ( 1 ) developed a method for meth:. io1 in which the methanol is oxidized to formaldehyde, which is then determined by this same colorimetric reaction. In endeavoring to determine terminal unsaturation by an indirect chemical method based on a formaldehyde determination, Bricker and Roberts ( 4 ) have pointed out that certain organic substances, such as benzene and acetone, inhibit the color formation of formaldehyde-chromotropic acid. I n order t o eliminate this interference, two color reactions for formaldehyde, Schiff's reagent and the potassium ferricyanidephenylhydrazine method of Schryver (9) were studied extensively. These methods were proved to be no better and in most respects inferior to the chromotropic acid reaction. Furthermore, the volumetric method using the optimum conditions with hydroxylamine hydrochloride (6) was investigated and was found to lack the specificity and sensitivity of colorimetric methods. Consequently, a thorough study of the chromotropic acid reaction was undertaken to improve the specificity and reliability of this reagent for formaldehyde in the presence of organic compounds. All previous methods using chromotropic acid as a formaldehyde reagent have developed the purple color by adding a definite volume of the solution to be analyzed to a solution of chromotropic acid. Sulfuric acid is then added and the resulting solution is heated for about 30 minutes in a boiling water bath. No positive interferences, except diacetyl ( 10) and possibly glycerylaldehyde, furfural, and some sugars ( 5 ) , have ever been reported.
However, if certain organic compounds are present with the formaldehyde, a less intense color is produced, This has been at.tributed to the fact that formaldehyde couples with many compounds in strong sulfuric acid and thereby prevents its color development with the reagent. Because there is no apparent color change when chromotropic acid and a formaldehyde solution are heated, i t has been assumed previously that there is no reaction between these two compounds unless sulfuric acid is present. I n this investigation it has been found that chromotropic acid does react with or retain formaldehyde when a solution is evaporated t o dryness. During this evaporation the volatile organic compounds which inhibit the purple dye formation are removed. When the sulfuric acid is added and the resulting solution is heated, the correct amount of the purple dye is produced. RECOMMENDED PROCEDURE
10 mg. of chromotropic acid (obtained from Weigh 100 Paragon Division of The hlatheson Company, Inc.) into a 30-nd. beaker. Add a definite volume of the solution to be analyzed, no more than 1 ml. in volume or 100 micrograms in formaldehyde content. If less than 1 ml. of solution is taken, add sufficient water to make 1 ml. Evaporate the solution to dryness on a low temperature hot plate or in an oil bath whose temperature does not exceed 200" C. and heat the residue for at lesst 5 minutes after the last traces of liquid have disappeared from the sides of the beaker. Allow to cool and then add 5 ml. of concentrated sulfuric acid. Heat the resulting solution in boiling water for 30 minutes. Cool and dilute to 50 ml. with water in a volumetric flask. Allow the diluted solution to reach room temperature and then measure the optical density against a reagent blank a t 570 mp. Several known quantities of formaldehyde are carried through this procedure in order to obtain a calibration curve. Because this curve is linear, the number of micrograms of formaldehyde
721
V O L U M E 22, NO. 5, M A Y 1 9 5 0 in an unknown sample is calculated by merely dividing the measured optical density by the density produced by 1 microgram of formaldehyde in the calibration curve.
Table 11. * Ratio
EXPERIMENT4L
10:1
I n one series of experiments, a constant amount of formaldeh de in 1 ml. of solution was added to various weights of reagent. d e s e data, shown in Table I, indicate that the optical density increased gradually as the weight of chromotropic acid was increased. There was, however, very little change in the optical density when a 100- or 150-mg. portion of reagent was used. Calibration curves with pure formaldehyde solutions were then run using 50, 100, and 150 mg., respectively, of chromotropic acid for each determination. All these calibration curves were linear to a t least 100 micrograms of formaldehyde, whereas the older method (3) started to deviate from linearity above 60 micrograms. Although the calibration curves in which 100 or 150 mg. of reagent were used were practicallv superimposable, the curve obtained with 50 mg. had a slightly lower slope.
Table I.
Variation of Color Produced with Weight of Reagent Used
Formaldehyde Taken
Chromotropic Acid T a k e n
Mo.
Mg.
0,050 0.050 0.050 0.050 0.050 0,050 0.050
0.050 0.050 0,050
10.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100,o 150.0
Extinction, Duplicate Determinations
0.070 0.285 0.321
0.375 0.380 0.387 0.392 0,397 0.402 0 I408
0.062 0.292 0.328 0.378 0.382 0.384 0.392 0,396 0 406 0.411
In view of these experiments, it was decided that better reproducibility and sensitivity could be expected if no less than 100 mg. of chromotropic acid were used for each determination. It w a s found t h a t the recovery of formaldehyde decreased if the amount of solution taken was much greater than 1 ml. Larger volumes than 1 ml. could be analyzed for formaldehyde by using proportionally larger quantities of reagent. Other acids in addition to sulfuric acid were used to develop a color after the drying operation. When sirupy phosphoric acid was used, a n orange-purple color was formed. Hydrochloric acid gave a pinkish purple color and acetic acid yielded a canary yellow. The optical densities a t 570 mg of these solutions were much lower in all cases than those obtained from an equal amount of formaldehyde when sulfuric acid was used. No further work was done with these acids hecause the sensitivity was so milch lower.
Formaldehyde Added
MQ.
All spectrophotometric readings were made on a Beckman Model DU spectrophotometer using a slit width of 0.04 mm. The first experiments using the recommrnded procedure with four 10-microgram quantities of formaldehyde per milliliter showed t h a t the reproducibility of the method was extremely good. Therefore, i t seemed likely t h a t a reproducible amount of formaldehyde was retained by the reagent during the evaporation. The temperature of the hot plate or oil bath during the evaporation should be such as not to cause spattering. Inasmuch as the temperatures of hot plates vary appreciably, the stability of the residues and the subsequent repfoducibility of the purple color were studied by using an oil bath which was maintained at various temperatures. It was proved that the dried residues did not undergo any change if they were allowed to stand for 20 minutes a t 170" C. Furthermore, the recovery of formaldehyde from mixtures of organic compounds was perfect when a n oil bath maintained at a temperature as high as 200" C. was used for the evaporation step in the recommended procedure. The amount of chromotropic acid necessary for a determination was determined in two ways.
Recovery of Formaldehyde
40:1 100: 1 100:1 200:1
10: 1
40:l 100:1
1OO:l 200:1 1000 : 1 4000 : 1 10,000: 1 10,000: 1
20,000:1
1000:l 4000 :1 10,000:l 10,000:1 20,000:1
Formaldehyde Found Old Recommended procedure (3) procedure Mg. 'MQ.
Acetaldehyde t o formaldehyde 0.050 0.0481 0.050 0.0461 0.050 0.010 0.010
0.0491 0.0481
0,0440
0,0448
0.0041 0.003s
0.009,
Benzaldehyde to formaldehyde 0.050 0.048r 0.050 0,0460 0.050 0.044, 0.0044 0.010 0.010 0.0036 Benzene to formaldehyde 0.050 0.046a 0.050 0,0381 0.050 0.0280 0.010 0,0032 0.010 0.0020 Pyridine to formaldehyde 0.050 0.0341 0.050 0,024,
0.0084
0.0499 0.049s 0,0490 0.0091 0.008a
0.0491 0.0491 0,0489 0.0098 0.009s 0,0508
0.050
0.015s
0,0508 0,0506
0.010 0.010
0.004s 0.0031
0,009, 0,0099
APPLICATIONS AND INTERFERENCES
It has been shown ( 9 ) t h a t n-propyl alcohol, n-amyl alcohol, methyl ethyl ketone, acetone, and other organic compounds inhibit the formaldehyde color development and thereby give low recoveries. This is also shown in Table 11, where this same procedure was used t o determine formaldehyde in the presence of acetaldehyde, benzene, pyridine, and benzaldehyde. With the procedure recommended in this paper, formaldehyde recoveries have been investigated in the presence of 20 different organic compounds. The results of a few of these studies are shown in Table 11. The recommended procedure has proved suitable for determining as little as 1 part of formaldehyde in the presence of 20,000 parte of chloroform, carbon tetrachloride, methanol, ethyl alcohol, n-butyl alcohol, isobutyl alcohol, eec-butyl alcohol, tertamyl alcohol, acetone, methyl ethyl ketone, pyridine, arid benzene. The recovery of formaldehyde in the presence of acetic acid, propionic acid, and benzyl alcohol is probably reliable to only 1 part in 1000 or 2000 of these compounds. With acetaldehyde or benzaldehyde present, the recovery of formaldehyde is reliable to only about 1 part in 100. Benzoic acid shows the greatest interference, b u t b y using small samples, the recovery of formaldehyde can be sssumed to be quantitative in the presence of ten times as much of this compound. The fact that benzoic acid is not as volatile as the other compounds studied undoubtedly accounts for its marked interference. When an attempt was made to determine formaldehyde in the presence of iodine, erratic results were obtained. There wm no apparent correlation of formaldehyde recovered to the amount of iodine present. However, formaldehyde can be determined very accurately by the recommended procedure in the presence of a t least 25 times as much iodine if the iodine is reduced with sodium sulfite before the sample is added to t h e chromotropic acid. The amount of sodium sulfite used is not critical; a 1000-fold exress did not appreciably effect the recovery. The cyclic formals, such as are present in safrol and piperonal, yield formaldehyde on heating with acid. This formaldehytfr of constitution can be determined by making use of the previously reported procedure (3). If the procedure reported in this paper is followed, these formals are removed by evaporation and only any free formaldehyde which was present originally is determined Henre, II combination of t h r two chromotropic acid proccdurcs
ANALYTICAL CHEMISTRY
722 will serve t o determine safrol or other similar compounds and formaldehyde in the presence of each other. Speck (10)has pointed out t h a t formaldehyde will interfere with the determination of diacetyl by his procedure. Samples of diacet yl when carried through the recommended procedure produce no color. Furthermore, diacetyl, at least in small concentrations, does not interfere with the determination of any free formaldehyde that may be present. Therefore, a combination of these procedures will serve to determine formaldehyde and diacetyl in the presence of each other. Compounds which are not readily volatile a t 170" C. may still interfere with the formaldehyde reaction. However, the usefulness of this reagent has been greatly extended with this modified procedure. This procedure gives a linear calibration line and thereby eliminates the necessity of using a calibration graph to determine the amount of'formaldehyde corresponding t o a given optical density.
ACKNOWLEDGMENT
The authors gratefully acknowledge the financial assistance of the grant made by E. I. du Pont de Nemours & Company to sponsor fundamental chemical research at Princeton University. LITERATURE CITED
Boos, R. N., ANAL.CAEM.,20, 964 (1948). Boyd, 31. J., and Logan, M. A , , J . B i d . Chem.. 146, 279 (1942). Bricker, C. E., and Johnson, H. R., IXD.ESG.CAEM.,ANAL.ED., 17,400 (1945). Bricker, C. E., and Roberts, K. H., ANAL.CHEM.,21, 1331 (1949). Eegriwe, E., Z. anal. Chem.. 110, 22 (1937). Huckabay, W. B., Newton, C . J., and Metler, A. V., ANAL. CHEM..19, 838 (1947). Kleinert, T.. and Srepel, E., Mikrochemie uer. Mikrochim. Arta. 33, 328 (1948). MacFayden, D. A., J . Bid. Chem., 158, 107 (1945). Schryver, S. B., Proc. R o g . Soc. (London), 82B,226 (1909). Speck, J. C.. Jr., ANAL. C H E M .20, . 647 (1948). R E C E I V E DSovember 28. 1949.
Determination of Benzylpenicillin DOROTHY J. HISCOX Department of Wational Health and Welfare, Ottawa, Ontario, Canada 1946, Page and Robinson (6) published a colorimetric IN (N1-naphthyl) method for the determination of benzylpenicillin, using ethylenediamine dihydrochloride a s a color reagent. By this method the benzene ring of the penicillin was nitrated, and the nitro compound was reduced, then diazotized and coupled with the color reagent in the presence of ethyl alcohol. A violet color showing maximum absorption a t a wave length of 560 mp was produced. This method did not give reproducible results when tested in this laboratory. During investigational work, two reasons for the failure of this method were found: An excess of sodium nitrite reacts with the color reagent, and under the conditions outlined by the arithors, nitration was not complete. This is also true of other methods for the determination of benzylpenicillin which are based upon the nitration of the benzene ring (f-S). Under none of the conditions used in these methods could reproducible results be obtained when tested colorimetrically with N( 1-naphthyl) ethylenediamine dihydrochloride. The error did not lie in the color development, as there was no significant variation in the color developed in numerous aliquots taken from single nitrations. Nitration was more complete with smaller esmples of penicillin. For routine work the simplest conditions for nitration would be to heat the sample with nitration mixture in a test tube in boiling water. It was decided to see if complete nitration could be effected under these conditions. Surprisingly, i t was found that nitration was more complete when smaller aliquots of nitration mixtures were used. When the nitration mixture was broken down into its two components, it was shown that this result wa3 due to the decrease in the amount of sulfuric acid present with the smaller aliquots. Typical results are shown in Table I. With sample 1, as the amount of sulfuric acid increased, the color produced decreased. This could be due to the decrease in the concentration of potassium nitrate present in the mixture. However, samples 2 and 3 show that i t was due to the amount of sulfuric acid present. With these samples, different amounts of the same nitration
mixture were used, but the concentration of the potassium nitrate was unchanged. Bitration was more complete with the smaller amount of nitration mixture-that is, with less sulfuric arid present . It was found that, when a sample of 0.5 mg. of penicillin or less is heated in boiling water for 2 hours with 0.5 ml. of a nitration mixture containing 60 grams of potassium nitrate in 100 ml. of concentrated sulfuric acid, nitration is complete. Under these conditions, reproducible results are obtained. The density of the color from an amount of aniline equivalent to 0.5 mg. of benzylpenicillin, as determined in a Beckman quartz spectrophotometer a t 560 mfi, was 1.20 and 1.30. The density of the color from 0.5 mg. of benzylpenicillin was 1.19, 1.23, and 1.33. Although this cannot be taken as an absolute comparison, it is a good indication that nitration by the author's procedure is complete. When such a small amount of nitration mixture is used, it is necessary to add more sulfuric acid after nitration is completed before the color is developed to ensure its completp development. Heat from 0.1 to 0.25 nig. of penicillin with 0.5 ml. of nitration mixture (60 grams of potassium nitrate in 100 ml. of concentrated sulfuric acid) in a 25 X 100 mm. test tube in boiling water for 2 hours. Add 5 ml. of water and 0.2 gram of granular zinc and heat an additional 15 minutes. Transfer to a 25-ml. volunwtrib flask, rinsing the tube with two 3-ml. portions of water and decanting the liquid from the zinc residue. Add l ml. of ronren-
Table I.
Effect of Sulfuric Acid on Nitration of Benzylpenicillin
Sample
KSOr, G.
HzSO4. MI.
1
0 40
0 40 0 40
1 0 1 5 2 0
2
0 40 0 80
1 0 2 0
3
0 25 0 50
0 5 1 0
70Absorption 57 9 17 4
51 9 16
c(
3 2 74 7 27 3
73 8 24 2
89 9 75 4
'10 2 74 0
4 7
